This invention relates to methods for determining the concentration of substances in biological fluids. More particularly it relates to such methods wherein magnetically responsive composite microparticles having a permeable, solid, water-insoluble matrix comprising a proteinaceous material, a polysaccharide, a polyurethane or mixtures thereof are employed.
Techniques useful in determining concentrations of substances in biological fluids, such as serum or urine, are known. The most common of these techniques are in vitro assay procedures. Frequently they involve competitive binding of the substance whose concentration is to be determined, and a radioactively labeled analog thereof, to binding sites on a specific receptor (usually a protein or antibody). The portion of the substance bound to the receptor is then separated from the portion not bound to the receptor and counted for radioactivity. The concentration of the substance is then determined by comparing the counted radioactivity with a standard curve.
Techniques employed to separate the bound from the unbound fractions include, for example, differential migration of the bound and free fractions (e.g., by electrophoresis, chromatoelectrophoresis, gel filtration, etc.); chemical precipitation of the bound or free fraction (e.g., by means of organic solvents, salts, acids, etc.); immunological precipitation of the bound fraction (e.g., by the double antibody method); adsorption of the bound or free fraction onto selective sorbing media (e.g., charcoal, silicates, resins, etc.).
Some of these techniques require rather complicated procedures in order to bring about separation of the bound from the free fractions. Such procedures reduce operator efficiency and increase the cost of the tests. As a result they are not suited to large scale operations. Other of these techniques do not adequately distinguish between the bound and free fractions and therefore lack the accuracy preferred in in vitro testing. Still other of these techniques can only be utilized in a limited number of tests.
Recently attempts have been made to simplify in vitro testing for substances present in biological fluids. For example, U.S. Pat. No. 3,933,997 discloses a process and composition useful in determining concentrations of digoxin in body fluids. The composition consists of anti-digoxin antibodies covalently bonded to an intermediate silane which is in turn coupled to magnetically responsive particles. The particles can be magnetically separated from a test solution.
Antibodies used in such composition are, therefore, coupled to the surface of the particles thereby limiting the amount of antibody present thereon. This has the further effect of limiting the binding capacity of the compositions. Additionally, when such compositions are prepared from anti-sera having low titer certain particles may not have any antibody bound thereto. Additionally, the process of covalently bonding the antibody to the intermediate silane must be carefully controlled so that the complexing ability is not lost. Moreover, this composition is only useful in determining concentrations of digoxin.
U.S. Pat. No. 3,970,518 discloses small magnetic particles whose surface is coated with a layer of antibody. Such coated magnetic particles are useful in sorting out and separating tiny select viruses, bacteria and other cells from multi-cell, bacteria or virus populations. The amount of antibody in such compositions available for sorting and separating is also limited by the available external surface area of the magnetic material.
The process of the present invention overcomes these deficiencies of the prior art. It employs compositions wherein the permeable, solid, water-insoluble matrix of the microparticle itself is capable of sorbing substances present in biological fluids (e.g., drugs, hormones, vitamins, proteins, enzymes). Additionally, the sorptive properties of the microparticles useful in the process of the invention are not lost during their preparation. Still further, the microparticles useful in the process of the present invention have a substantially greater binding capacity than do compositions employed in prior art processes. This is because the microparticles useful in the present invention are permeable thereby allowing biological fluids to penetrate into the interior of the particle and be sorbed therein. As a result the binding capacity of particles useful in the present invention is not limited by the amount of the external surface area of the particle. As a result the process of the present invention provides a more accurate diagnostic test than do prior art processes. Furthermore, the process of the present invention is useful in a wide variety of in vitro diagnostic tests.
The process of the present invention is, therefore, useful in determining the concentration of a variety of substances in biological fluids in a rapid, efficient and accurate manner. Thus it may be employed to measure the amount of drugs (e.g., digoxin and antibiotics), hormones (e.g., thyroxine), vitamines (e.g., vitamin B.sub.12), proteins, enzymes, etc. Because the particles useful in the invention are magnetically responsive they are rapidly and efficiently separable from the test solution. Thus the process is simple to use and eliminates the necessity to employ complicated and expensive separation techniques. Consequently it is particularly useful in large scale testing wherein a number of separate samples are tested at the same time. Moreover, the process of the invention may be designed to test for a specific substance or a variety of substances.